CN112010975B - LAG3 binding fragments and uses thereof - Google Patents

Abstract

The present invention provides an antibody or antigen binding fragment that binds to lymphocyte activation gene 3(LAG3) and inhibits, interferes with, or neutralizes at least one activity thereof. The anti-LAG3 antibody or antigen-binding fragment can be used to treat a disease, such as cancer.

Description

LAG3 binding fragments and uses thereof

Technical Field

The invention relates to an antibody or antigen-binding fragment thereof binding to lymphocyte activation gene 3(LAG3), and a preparation method and application thereof in preparing a pharmaceutical composition and treating diseases.

Background

LAG3(lymphocyte activation gene 3, LAG3, CD223) belongs to the immunoglobulin superfamily, and is an immune checkpoint receptor protein, primarily expressed on activated T cells, NK cells, B cells and plasma cell dendritic cells. LAG3 down regulates T cell activity primarily through binding to ligand MHC II molecules. Inhibition of LAG-3 allows T cells to regain cytotoxicity, thereby enhancing the killing effect on tumors. And the function of regulating T cells to inhibit immune response can be reduced by inhibiting LAG-3.

Clinical data also show that in a variety of cancer types, such as melanoma, colon cancer, breast cancer, etc., Tumor Infiltrating Lymphocytes (TILs) express LAG-3, which correlates with the clinical features of cancer cell aggressiveness. Blocking LAG-3 reverses the above inhibitory effects, restoring CD8+ T cell proliferation and activity, and reducing the number of regulatory T cells; but also increases the sensitivity of the T cell immune response. Meanwhile, PD-1 is blocked, so that the immune response can be enhanced synergistically, and tumors are inhibited. Then LAG inhibitors may be administered in combination with or may be potentiated by PD1 inhibitors.

Disclosure of Invention

The various embodiments described throughout this disclosure with respect to VL domains, VH domains, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 may each be practiced alone or in any combination.

In one aspect of the invention, there is provided an antibody or antigen-binding fragment thereof comprising:

in some embodiments, the antibody or antigen-binding fragment thereof comprises any one combination of a VL domain and a VH domain as described in (1) - (2) below:

(1) a VL domain comprising the amino acid sequence of LCDR1 shown in SEQ ID NO. 5, the amino acid sequence of LCDR2 shown in SEQ ID NO. 6 and the amino acid sequence of LCDR3 shown in SEQ ID NO. 11, and a VH domain comprising the amino acid sequence of HCDR1 shown in SEQ ID NO. 12, the amino acid sequence of HCDR2 shown in SEQ ID NO. 13 and the amino acid sequence of HCDR3 shown in SEQ ID NO. 14;

(2) a VL domain comprising the amino acid sequence of LCDR1 shown in SEQ ID NO. 5, the amino acid sequence of LCDR2 shown in SEQ ID NO. 6 and the amino acid sequence of LCDR3 shown in SEQ ID NO. 7, and a VH domain comprising the amino acid sequence of HCDR1 shown in SEQ ID NO. 8, the amino acid sequence of HCDR2 shown in SEQ ID NO. 9 and the amino acid sequence of HCDR3 shown in SEQ ID NO. 10;

in some embodiments, the antibody or antigen-binding fragment thereof comprises any one combination of a VL domain and a VH domain as described in (1) - (2) below:

(1) a VL domain of the amino acid sequence shown in SEQ ID NO. 3 and a VH domain of the amino acid sequence shown in SEQ ID NO. 4;

(2) the VL domain of the amino acid sequence shown in SEQ ID NO. 1 and the VH domain of the amino acid sequence shown in SEQ ID NO. 2.

In one aspect of the invention, the antibody or antigen binding fragment thereof comprises a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a Fab, Fab ', F (ab') 2, Fv, scFv, or dsFv fragment.

In one aspect of the invention, an antibody or antigen binding fragment thereof of the invention binds to lymphocyte activation gene 3(LAG 3).

In one aspect of the invention, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO. 15.

In one aspect of the invention, the antibody or antigen-binding fragment thereof comprises a light chain constant region of the amino acid sequence set forth in SEQ ID NO. 18.

In one aspect of the invention, the invention relates to a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to the invention.

In one aspect of the invention, the invention relates to a vector expressing an antibody or antigen-binding fragment thereof according to the invention.

In one aspect of the invention, the invention relates to a cell that expresses an antibody or antigen-binding fragment thereof of the invention.

In one aspect of the invention, the invention relates to a method of treating cancer using an antibody or antigen-binding fragment thereof of the invention, comprising administering the antibody or antigen-binding fragment of the invention to a subject in need thereof. Preferably, the cancer is selected from lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, various sarcomas, cell tumors, and leukemia.

In one aspect of the invention, the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention for the treatment of cancer or for the preparation of a pharmaceutical composition for the treatment of cancer. Preferably, the cancer is selected from lung cancer, gastric cancer, esophageal cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, and leukemia.

In one aspect of the invention, there is provided a composition comprising an antibody or antigen-binding fragment thereof of the invention that specifically binds lymphocyte activation gene 3(LAG3), and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvent, dispersant, additive, plasticizer and medicinal auxiliary material.

In some embodiments, the composition may further comprise an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent, an immunotherapeutic agent, or a hormonal therapeutic agent. The antibody or antigen-binding fragment can be administered in combination with other therapeutic agents to enhance the therapeutic effect.

In some embodiments, the additional therapeutic agent is an immunotherapeutic agent, preferably the immunotherapeutic agent is an anti-PD 1 antibody or antigen binding fragment thereof, preferably the anti-PD 1 antibody is Opdivo.

In some embodiments, the subject is a mammal, e.g., a human. In other embodiments, the subject is an isolated cell, tissue, or organ, e.g., a cell cultured ex vivo.

In some embodiments, the "enhancing the therapeutic effect" refers to enhancing the therapeutic effect of another therapeutic agent or therapy. The antibodies or antigen binding fragments provided herein can be administered alone or in combination with other therapeutic agents or therapies. In some embodiments, the other therapeutic agent or therapy comprises a chemotherapeutic agent, an immunotherapeutic agent, a hormonal therapeutic agent, radiation therapy, surgical therapy.

The antibodies or antigen binding fragments provided by the invention have one or more of the following: enhanced LAG3 protein binding capacity, enhanced capacity to stimulate IFN-gamma secretion from T cells, enhanced capacity to promote T cell proliferation, enhanced capacity to stimulate IFN-gamma secretion from T cells when combined with PD1 antibody, and enhanced capacity to promote T cell proliferation when combined with PD1 antibody. In some embodiments, the control antibody is an antibody that specifically binds LAG3 protein as found in the prior art, e.g., such an antibody can be searched by a database. In some embodiments, the control antibody is a LAG3-BMS antibody.

Drawings

Figure 1A shows serum titers after quadruplicate immunization of mice.

Figure 1B shows serum titers after seven immunizations of mice.

FIG. 2 shows the sensitivity of Elisa to detect anti-LAG3 binding to LAG 3.

FIG. 3 shows FACS detection of binding of anti-LAG3 antibody on 293T-LAG3 cells.

FIG. 4 shows FACS detection of blocking activity of anti-LAG3 antibody at the cellular level.

FIG. 5 shows the mixed lymphocyte reaction assay for anti-LAG3 antibody in vitro cellular activity.

FIG. 6 shows the sensitivity of Elisa to detect anti-LAG3 antibody binding to LAG 3.

FIG. 7 shows IFN- γ secretion from anti-LAG3 antibody alone and in combination.

FIG. 8 shows the T cell proliferation activity of anti-LAG3 antibody alone and in combination with opsivo.

Fig. 9A shows the affinity of LAG3-BMS for LAG 3.

Fig. 9B shows the affinity of LAG3Q34-CA46 for LAG 3.

Fig. 9C shows the affinity of LAG3Q817-CA108.1 to LAG 3.

FIG. 10 shows the mean time course of the antibody administered to each cynomolgus monkey.

FIG. 11 shows the anti-LAG3 antibody immunogenicity assay.

Figure 12 shows growth curves for each group of tumor volumes in the MC38 mouse colon cancer model.

Fig. 13 shows a comparison of tumor weights in each experimental group after the end of dosing.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The described embodiments are only some, but not all embodiments of the invention. It is to be understood that the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compositions of the present invention are utilized, and are not intended to limit the scope of what the invention pertains. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 production of anti-LAG3 monoclonal antibody

Example 1.1 immunization of mice

A Boan-hMab safe human antibody transgenic mouse is immunized by emulsifying LAG3 (Chinesia, Yi Qiao, 16498-H08H) and Freund's adjuvant. This time, 20 mice were co-immunized, to five or seven immunizations, respectively. 5 mice with higher serum titers were selected for boosting, and 3 days later the mice were sacrificed and spleens were removed for subsequent experiments.

Example 1.2 phage library Generation

Taking spleen cells of an immune mouse, adding Trizol (Thermo Scientific, catalog number 15596-026), adding 1/5 volumes of chloroform after full lysis, fully mixing, standing at room temperature for 20min, centrifuging at 4 ℃ 12000rpm for 20min, taking an upper layer aqueous solution, adding isopropanol with the same volume, standing at room temperature for 20min, centrifuging at 4 ℃ 12000rpm for 20min, discarding a supernatant aqueous solution, adding 75% ethanol for washing twice, centrifuging at 4 ℃ 12000rpm for 5min, discarding an aqueous solution, keeping a precipitate, air-drying at room temperature, adding DEPC water for re-suspending the precipitate to obtain RNA, and carrying out reverse transcription on the RNA into cDNA by using a Roche Applied Science, catalog number 4897030001) according to the specification. Phage library establishment procedures reference Carlos f. barbas III, Phage display: the method described in A laboratory manual was performed by obtaining the variable regions of heavy and light chains from cDNA by PCR, and then combining the heavy and light chainsObtaining ScFv by overlap extension PCR method from the variable region of the light chain, digesting ScFv with SfiI (NEB, catalog number R0123L) for 50 ℃ 5h, ligating the digested ScFv (single chain Fv) with plasmid pCOMB3x (Stratagene), electrotransfecting the ligated product into competent cells of Escherichia coli TG1, culturing at 37 ℃ 200rpm for 1h, adding 75mL of 2YT medium (with ampicillin and glucose added), culturing at 37 ℃ 200rpm for 1.5h, taking 2mL for measurement of Escherichia coli TG 600, adding previously prepared cage at a ratio of 1:20, standing at 37 ℃ for 30min, centrifuging at 4000rpm to collect bacterial pellet, culturing at 30 ℃ 200rpm with 100mL of 2YT medium (with ampicillin and kanamycin added), next day, centrifuging at 12000rpm for 10min, adding 4% PEG8000 and 3% NaCl, 1201 h, taking out the OD 10min, adding the OD 30 rpm, resuspending and adding 2 medium overnight, adding 7% DMSO, and freezing for later use. Phage library LAG3Q8, library capacity 5X 10, established with mouse accession LAG3Q8 ⁸ (ii) a Phage library LAG3Q 17, 8X 10 library capacity, established with LAG3Q 17 mouse ⁸ (ii) a Phage library LAG3Q3, library capacity 6.4 × 10, established with mouse number LAG3Q3 ⁸ (ii) a Phage library LAG3Q 4, library capacity 6X 10, established with mouse accession number LAG3Q 4 ⁸ 。

Example 1.3 phage selection

1. Plate screening, plate coating with LAG3 protein (16498-H08H, Protovacua, supra) at 1. mu.g/well, standing overnight at 4 deg.C, blocking the plate with 2% BSA the next day for 1H, and adding phage library (2X 10) ¹² ) After 2h incubation, the specifically bound LAG3 phages were eluted with an Elution Buffer (pH 2.2) after 4-10 washes.

2. Magnetic bead screening, in which LAG3-Fc protein (16498-H02H, Chinesia, Yinqiao) is biotinylated according to the conventional procedure (molar ratio of the added LAG3-Fc protein to biotin 1:2), and then is combined with Thermo magnetic beads (Invitrogen Dynabeads M-280Streptavidin, 00355871), and then incubated with a phage library, and after washing for 4-10 times, the specifically bound phage of LAG3 are eluted by Elution Buffer (pH 2.2).

Clone sources are as in table 1.

TABLE 1 sources of Positive clones

Clone ID	Source bank	Mouse of origin	Elutriation method
				LAG3Q34-CA46-IgG4	Q3+Q4	LAG3Q3，LAG3Q4	Plate screening
LAG3Q34-CA242-IgG4	Q3+Q4	LAG3Q3，LAG3Q4	Plate screening
				LAG3Q817-CA108-IgG4	Q8+Q17	LAG3Q8，LAG3Q17	Plate screening
LAG3Q817-CA116-IgG4	Q8+Q17	LAG3Q8，LAG3Q17	Plate screening
				LAG3Q817-CA139-IgG4	Q8+Q17	LAG3Q8，LAG3Q17	Plate screening
LAG3Q817-CA141-IgG4	Q8+Q17	LAG3Q8，LAG3Q17	Plate screening
				LAG3Q2526-CA152-IgG4	Q25+Q26	LAG3Q25，LAG3Q26	Plate screening
LAG3Q817-CA265-IgG4	Q8+Q17	LAG3Q8，LAG3Q17	Plate screening
				LAG3Q817-CA271-IgG4	Q8+Q17	LAG3Q8，LAG3Q17	Plate screening

EXAMPLE 1.4 molecular construction and production of antibodies

Clones LAG3Q34-CA46, LAG3Q2526-CA152, LAG3Q817-CA108, CA116, CA139, CA141, CA265, CA271 were sent to Invitrogen Biotechnology Ltd for sequencing. The amino acid sequence of each clone is shown in Table 2.

TABLE 2 cloned amino acid sequences with blocking Activity

The variable region sequence of the antibody was amplified by conventional PCR (2 XEasypfu PCR Supermix manufacturer: Transgen cargo No.: AS211 batch No. # L11228), a linker of the signal peptide and the variable region gene was obtained by overlap extension PCR, homologous recombination (CloneseIIOne Step Cloning Kit manufacturer: Vazyme cargo No.: C112-01 batch No. # TE222B8) and the like, a nucleotide sequence fragment encoding the VH domain was inserted into a vector pC3.4 (Life Technology) carrying a nucleotide sequence encoding the antibody heavy chain constant region amino acid sequence (SEQ ID NO:15), a nucleotide sequence fragment encoding the VL domain was inserted into a vector pC3.4 (LiDNAtechnology) carrying a nucleotide sequence encoding the antibody light chain constant region amino acid sequence (SEQ ID NO:18), transfected into HEK293 cells at 37 ℃ C. 8% CO2 rpm, and transiently expressed on a shaker for 7 days by ProteA affinity chromatography, the anti-LAG3 antibody was obtained by purification and the antibody concentration was determined by UV280 binding extinction coefficient.

Control antibody production: the amino acid sequence of the Baishiqianbao LAG3 antibody Relatimab was determined by IMGT data and patent CN201580006297, the vector pCDNA3.4 was inserted after the whole gene synthesis and expressed by HEK293 cells, and the produced antibody was named LAG3-BMS, the heavy chain sequence of which is shown in SEQ ID NO. 16 and the light chain sequence of which is shown in SEQ ID NO. 17.

Example 1.5 Elisa binding of detection antibodies to LAG3 protein

Coating protein LAG3(16498-H08H, Hovenia) with different concentrations (3.2. mu.g/ml, 0.8. mu.g/ml, 0.2. mu.g/ml, 0.05. mu.g/ml, 0.0125. mu.g/ml, 0.003125. mu.g/ml, 0.00078125. mu.g/ml, 0. mu.g/ml), 100. mu.l/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100 μ l of each candidate antibody of 1 μ g/ml into each well, and incubating at 37 ℃ for 1 h; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIG. 2 and Table 3.

As shown in table 3, the candidate antibody LAG3Q34-CA46 bound LAG3 protein with an EC50 value of 0.01979, which is significantly lower than the EC50 value 0.04932 of the control LAG3-BMS, indicating that the candidate antibody has better antigen binding capacity than the control LAG 3-BMS.

As shown in table 3, the EC50 value of the binding of the candidate antibody LAG3Q817-CA108 to LAG3 protein was 0.02261, which is significantly lower than the EC50 value 0.04932 of the control LAG3-BMS, indicating that the antigen binding ability of the candidate antibody is better than that of the control LAG 3-BMS.

The candidate antibodies LAG3Q34-CA46 and LAG3Q817-CA108 are predicted to have stronger targeting and binding properties on LAG3 protein compared with a control group LAG3-BMS, and can achieve better pharmaceutical effects.

TABLE 3 Elisa detection anti-LAG3 antibody and LAG3 binding sensitivity data

Name of antibody	EC50(μg/mL)	Name of antibody	EC50(μg/mL)
				LAG3Q34-CA46	0.01979	LAG3Q34-CA242	0.01731
LAG3Q817-CA108	0.02261	LAG3Q817-CA271	0.03389
				LAG3Q817-CA116	0.02849	LAG3Q817-CA265	0.02293
LAG3Q817-CA139	0.05634	LAG3Q2526-CA152	0.2639
				LAG3Q817-CA141	0.06501	LAG3-BMS	0.04932

Example 1.6 flow cytometry detection of the binding Capacity of anti-LAG3 antibody to 293T-LAG3 in cells

To a 96-well round bottom plate, 50. mu.L of 293T-LAG3 cells (cell number 50000/well) were added, each antibody was diluted with a FACS buffer (sterile PBS, 0.2% BSA) gradient, added to the 96-well round bottom plate at 50. mu.L/well, and incubated at 4 ℃ for 1 h. After centrifugation at 2000rpm for 3min, the supernatant was discarded, washed 2 times with FACS buffer, 100. mu.L/well fluorescent secondary antibody (Southern Biotech, 2040-09) was added to a final concentration of 1. mu.g/ml, incubated at 4 ℃ for 1h, centrifuged at 2000rpm for 3min, discarded, washed 2 times with FACS buffer, resuspended at 100. mu.L/well FACS buffer, and examined by flow cytometry (Exson, Novocyte 2060). The results are shown in FIG. 3.

Example 1.7 FACS detection of anti-LAG3 antibody at cellular level blocks the Activity of LAG3 protein binding to Daudi cells

Transferring the sample and LAG3-mFc (1 μ g/ml) (Poppuss, LA3-H52Aa) into a 96 shallow well plate according to the ratio of (50+50) μ l/w, mixing uniformly, incubating at room temperature for 30min, and then culturing the Daudi cells according to the ratio of 1 × 10 ⁵ And/w transferring into a 96-hole shallow-hole plate, centrifuging at 2500rpm for 3min, then discarding the supernatant, transferring into a mixed 100 mu l antibody sample, mixing uniformly, and standing for 1h at 4 ℃. Centrifuging at 2500rpm for 3min, washing the cells with DPBS containing 0.2% BSA 2 times, adding 100. mu.l/w fluorescenceA secondary antibody (1:200) (Biolegend, 407108) was mixed well and left to stand at 4 ℃ for 1 hour. After removal, centrifugation was carried out at 2500rpm for 3min, and the cells were washed 2 times with DPBS containing 0.2% BSA, resuspended in 50. mu.l/w of DPBS containing 0.2% BSA and assayed by flow cytometry. The inhibition ratio ═ MFI (buffer) -MFI (sample))/MFI (antibody at concentration 0) × 100. The results are shown in FIG. 4.

Example 1.8 Mixed lymphokines assay to test the in vitro function of anti-LAG3 antibody

Resuscitating CD4+ T cells, centrifuging at 1000rpm for 5min, resuspending the cells in complete medium and counting, adjusting the cell density to 1X 10 with complete medium ⁶ one/mL. CD4+ T cells were added to a 96 well cell culture plate at 100. mu.L per well, resulting in 100000 viable cells per well, cultured at 37 ℃ in a 5% CO2 incubator for 5-7 days. Diluting 200 Xcapture antibody (anti-IL-2 antibody or anti-IFN-gamma antibody) to 1X with coating buffer solution, adding 100 μ L to each well of 96-well enzyme label plate, and incubating overnight at 2-8 ℃; the capture antibody in the plate was discarded, and the 96-well plate was washed 4 times with 200. mu.L of PBST (PBS + 0.05% Tween 20); add 200. mu.L of 1 × Assay dilution A per well, incubate 1 hour at room temperature (500rpm), wash 96-well microplate 4 times with 200. mu.L PBST; diluting the supernatant to be detected to a proper proportion by taking 1 xAssay dilution A, mixing uniformly, and adding 100 mu L of diluted supernatant to an ELISA plate coated with IFN-gamma or IL-2 capture antibody. Standards were diluted simultaneously, 100. mu.L of each well was added to the microplate and incubated for 2 hours (500rpm) at room temperature. Washing 96-well enzyme label plate with 200 μ L PBST for 4 times; 200 Xthe detection antibody (biotin-labeled anti-IL-2 antibody or biotin-labeled anti-IFN-. gamma.antibody) was diluted 1X with 1 XSasy reagent A, 100. mu.L was added to a 96-well plate, and incubated at room temperature for 1 hour (500 rpm). Washing 96-well enzyme-linked plate 4 times with 200 μ L PBST; avidin-labeled horseradish peroxidase (Avidin-HRP) was diluted 1000-fold with 1 × Assay dilution A, and 1000. mu.L of the diluted solution was pipetted into a 96-well plate and incubated at room temperature for 30 minutes. Washing 96-well enzyme-linked plate 5 times with 200 μ L PBST; adding 100 μ L of TMB developing solution into 96-well plate, incubating at room temperature for 10-20 min, and adding 50 μ L of stop solution (2M H) ₂ SO ₄ ) (ii) a The absorbance of light at a wavelength of 450nm (OD450) was measured with a microplate reader. The results are shown in FIG. 5.

Example 2 candidate antibody characterization

Example 2.1 LAG3Q817-CA108 molecular engineering

The heavy chain of CA108 is analyzed to find that the heavy chain contains N sugar sites, a primer is designed to mutate N into K, and the obtained new clone is marked as CA 108.1. The sequences are shown in Table 4.

TABLE 4 LAG3Q817-CA108 molecular engineering sequences

Using ExpicHO ^TM Expression System Kit (Thermofisiher Scientific, cat # A29133) transient transfection experiments were performed to investigate the transient Expression levels of each antibody. The respective antibody transient expression levels are shown in Table 5.

TABLE 5 Table of the transient expression yields of the antibodies

Name of antibody	Transient expression yield (mg/L)	Size heterogeneity (SEC-HPLC)
			LAG3-BMS	172.5	99.14％
LAG3Q817-CA108.1	505	99.55％
			LAG3Q34-CA46	144	99.49％

As can be seen from the expression amount of transient transfection, the expression amount of LAG3Q817-CA108.1 is the highest, which indicates that the antibody has better production feasibility.

Example 2.2 Elisa detection of binding of candidate antibodies to LAG3 protein

Coating protein LAG3(16498-H08H, Protoyotavia) at different concentrations (4. mu.g/ml, 1. mu.g/ml, 0.25. mu.g/ml, 0.0625. mu.g/ml, 0.015625. mu.g/ml, 0.0039063. mu.g/ml, 0.0009766. mu.g/ml, 0. mu.g/ml), 100. mu.l/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100 μ l of each candidate antibody at a concentration of 1ug/ml per well, and incubating at 37 deg.C for 1 h; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIG. 6 and Table 6.

TABLE 6 Elisa detection sensitivity of anti-LAG3 antibody binding to LAG3

Name of antibody	EC50(μg/mL)	Name of antibody	EC50(μg/mL)
				LAG3Q817-CA108.1	0.026	LAG3-BMS	0.032
LAG3Q34-CA46	0.022	/	/

As shown in table 6, the candidate antibody LAG3Q34-CA46 bound LAG3 protein with an EC50 value of 0.022, which is lower than the EC50 value of 0.032 of the control LAG3-BMS, indicating that the antigen binding ability of the candidate antibody is better than that of the control LAG 3-BMS.

As shown in table 6, the EC50 value of the binding of the candidate antibody LAG3Q817-CA108.1 to LAG3 protein was 0.026, which is lower than the EC50 value of 0.032 of the control LAG3-BMS, indicating that the antigen binding ability of the candidate antibody was better than the control LAG 3-BMS.

The candidate antibodies LAG3Q34-CA46 and LAG3Q817-CA108.1 are predicted to have stronger targeting and binding performance on LAG3 protein and achieve better pharmaceutical effect compared with the control group LAG 3-BMS.

Example 2.3 species Cross-reactivity and non-specific binding experiments

Different concentrations (4. mu.g/ml, 1. mu.g/ml, 0.25. mu.g/ml, 0.0625. mu.g/ml, 0.015625. mu.g/ml, 0.0039063. mu.g/ml, 0.0009766g/ml, 0. mu.g/ml) of protein were coated with CBS coating solution (pH9.6 carbonic acid solution): murine LAG3(53069-M08H, yinqiao, shenzhou), monkey LAG3 protein (LA3-C5252), human LAG3 protein (16498-H08H, yinqiao), and CD4(LE3-H5228, septosia) 100 μ l/well 4 degrees overnight; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100 μ l of each candidate antibody of 2 μ g/ml into each well, and incubating at 37 ℃ for 1 h; goat anti-human IgG/HRP (anti-fab secondary antibody for monkey LAG3 detection) was then added, incubated at 37 ℃ for 1h, developed for 10min, and OD450 read on a microplate reader. The results showed that each antibody had no cross-reactivity with CD4 protein and bound to monkey LAG3 protein to varying degrees. The results are shown in Table 7.

TABLE 7 binding of each antibody to LAG3 protein of different species and CD4 protein Cross-reactivity

Example 2.4 Mixed lymphokines test of the ability of candidate antibodies to stimulate IFN- γ secretion and promote T cell proliferation

Resuscitating CD4+ T cells, centrifuging at 1000rpm for 5min, resuspending the cells in complete medium and counting, adjusting the cell density to 1X 10 with complete medium ⁶ one/mL. CD4+ T cells were added to a 96 well cell culture plate at 100. mu.L per well, resulting in 100000 viable cells per well, cultured at 37 ℃ in a 5% CO2 incubator for 5-7 days. Diluting 200 Xcapture antibody (anti-IL-2 antibody or anti-IFN-gamma antibody) to 1X with coating buffer solution, adding 100 μ L to each well of 96-well enzyme label plate, and incubating overnight at 2-8 ℃; the capture antibody in the plate was discarded, and the 96-well plate was washed 4 times with 200. mu.L of PBST (PBS + 0.05% Tween 20); add 200. mu.L of 1 × Assay dilution A per well, incubate 1 hour at room temperature (500rpm), wash 96-well microplate 4 times with 200. mu.L PBST; diluting the supernatant to be detected to a proper proportion by taking 1 × Assay Diluent A, uniformly mixing, and adding 100 μ L of diluted supernatant to an ELISA plate coated with IFN-gamma or IL-2 capture antibody. Standards were diluted simultaneously, 100. mu.L per well was added to the microplate and incubated at room temperature for 2 hours (500 rpm). Washing 96-well enzyme-linked plate 4 times with 200 μ L PBST; 200 Xdetection antibody (biotin-labeled anti-IL-2 antibody or biotin-labeled anti-IFN-. gamma.antibody) was diluted 1X with 1 × Assay Diluent A, 100. mu.L was added to a 96-well plate, and incubated at room temperature for 1 hour (500 rpm). Washing 96-well enzyme-linked plate 4 times with 200 μ L PBST; avidin-labeled horseradish peroxidase (Avidin-HRP) was diluted 1000-fold with 1 × Assay dilution A, and 1000. mu.L of the diluted solution was pipetted into a 96-well plate and incubated at room temperature for 30 minutes. Washing 96-well enzyme-linked plate 5 times with 200 μ L PBST; adding 100 μ L of TMB developing solution into 96-well plate, incubating at room temperature for 10-20 min, and adding 50 μ L of stop solution (2M H) ₂ SO ₄ ) (ii) a The absorbance of light at a wavelength of 450nm (OD450) was measured with a microplate reader. The results of IFN-. gamma.secretion are shown in FIG. 7, and the results of T-cell proliferation are shown in FIG. 8.

As shown in FIG. 7, the combination of LAG3Q817-CA108.1 and Opdivo stimulated more IFN-. gamma.secretion from T cells than the combination of LAG3Q817-CA108.1 alone, LAG3-BMS alone, LAG3-BMS and Opdivo. The clinical combination of LAG3Q817-CA108.1 and Opdivo can stimulate immune response better to achieve the anti-tumor effect.

As can be seen in FIG. 8, LAG3Q817-CA108.1, whether used alone or in combination with Opdivo, has a greater capacity to stimulate T cell proliferation than LAG3-BMS alone, LAG3-BMS in combination with Opdivo. It is predicted that the vaccine may have better immune cell activation capacity than LAG3-BMS clinically in the future, thereby promoting stronger tumor killing of the immune system.

Example 2.5 comparison of the affinity of LAG3Q817-CA108.1, LAG3Q34-CA46 with LAG3-BMS

The proA sensor was immersed in PBST for more than 10 min. Each antibody was diluted to 3. mu.g/mL with PBST. LAG3 was diluted with PBST in five concentration gradients: 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78nM, PBST as blank control. Setting the detection step of each sample: each sample was cyclically tested after regeneration of the sensor with glycine buffer pH1.7, Baseline (100s), Loading (300s and 1nm), Baseline (100s), Association (300s), Dissociation (300 s). Data fitting was performed after the experiment was over using the 1:1 mode of the software Data analysts 9.0. The affinity data are shown in table 8 and fig. 9A-9C:

table 8.

RED96 detection of candidate antibody binding kinetics

Name of antibody	kon(1/Ms)	kdis(1/s)	KD(M)
				LAG3-BMS	6.98E+05	3.46E-04	4.96E-10
LAG3Q34-CA46	3.73E+05	5.12E-05	1.37E-10
				LAG3Q817-CA108.1	2.96E+05	1.07E-04	3.61E-10

Example 2.6 candidate antibody drug-delivery assessment

The antibody concentration was measured by the Elisa method using 3 cynomolgus monkeys for each antibody administered at 5mg/kg intravenously before administration, 1min, 15min, 30min, 6h, and 24h (Day 2), 72h (Day 4), 168h (Day 8), 240h (Day 11), 336h (Day 15), 408h (Day 18), 504h (Day 22), 576h (Day 25), and 672h (Day 29) after administration, and the results are shown in Table 9 and FIG. 10.

TABLE 9 mean pharmacokinetic parameters of the three antibodies after intravenous injection in each group of cynomolgus monkeys

After the cynomolgus monkey is intravenously injected with LAG3Q817-CA108.1, the blood concentration reaches the peak quickly and slowly, the concentration is still high at 28 days, and the T is _1/2 318.83h (about 13.3 days), AUC _(0-∞) 20340.53. mu.g/mL hour, CL 79.31 mL/hour; t after administration of LAG3-BMS _1/2 328.36h (about 13.7 days), AUC _(0-∞) 17981.41 μ g/mL hour. In summary, both LAG3Q817-CA108.1 and LAG3-BMS have very long halvesThe period of decline, from AUC _(0-∞) In the above view, LAG3Q817-CA108.1 has slightly better drug exposure.

Meanwhile, the immunogenicity of each antibody is detected by adopting an ELISA method, which comprises the following specific steps: coating the LAG3Q817-CA108.1, LAG3Q34-CA46 and LAG3-BMS with CBS coating solution (pH9.6 carbonic acid solution) at 0.125. mu.g/ml, 0.0625. mu.g/ml, 0.125. mu.g/ml and 100. mu.l/well overnight at 4 ℃; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100 Xserum into each hole, adding 100 μ l, and incubating at 37 deg.C for 1 h; then, 0.125. mu.g/ml, 0.0625. mu.g/ml, 0.125. mu.g/ml and 0.125. mu.g/ml of LAG3Q817-CA108.1-biotin, LAG3Q34-CA46-biotin and LAG3-BMS-biotin were added and incubated at 37 ℃ for 1 hour. Washing, adding streptomycin/HRP, and incubating at 37 ℃ for 1 h; after development for 10min, OD450 was read on the microplate reader. The results are shown in FIG. 11.

From the detection results, the immunogenicity of the tested antibody is low, and the immunogenicity of the LAG3Q817-CA108.1 is not increased in 0-28 days, which indicates low incidence rate of the anti-drug antibody in clinic.

Example 2.7 pharmacodynamic study of candidate antibodies in PD-1/LAG3 double KI HuGEMM mice for the treatment of subcutaneous mouse colon carcinoma MC38 model

A PD-1/LAG3 double KI HuGEMM mouse is inoculated with MC38 cells subcutaneously, and a mouse colon cancer subcutaneous transplantation tumor model is established. When the tumor grows to the average volume of 88mm ³ At that time, 40 mice were randomly divided into 5 groups of 8 mice according to tumor size. The experiment was carried out by intraperitoneal injection, twice weekly and three weeks for a normal saline control group, an Opdivo single drug group, an Opdivo and LAG3Q34-CA46 combined drug group, an Opdivo and LAG3Q817-CA108.1 combined drug group and an Opdivo and LAG3-BMS combined drug group. The evaluation of the efficacy was carried out on the basis of the relative tumor inhibition ratio (TGI), and the safety was evaluated on the basis of the change in body weight and the death of the animals. FIG. 12 is a growth curve of tumor volume for each group in the MC38 mouse colon cancer model. The combined use of LAG3Q817-CA108.1 or LAG3-BMS and Opdivo has better tumor inhibition effect than that of Opdivo, and the combined use of the LAG3Q817-CA108.1 and the LAG3-BMS is likely to improve the response rate of PD1 single drug on patients and bring better treatment effect to cancer patients.

Mice were sacrificed 21 days after dosing to examine tumor weight, and the tumor-inhibiting effect of the test antibodies was compared from tumor weight as shown in fig. 13.

In the experiment, the tumor-bearing mice have good tolerance to each tested antibody under the test dosage, and no obvious drug toxicity is observed. Opdivo had a slight anti-tumor growth effect on the PD-1/LAG3 double KI HuGEMM mouse subcutaneous colon cancer MC38 model at a dose of 2 mg/kg. When Opdivo was treated in combination with LAG3Q817-CA108.1 or LAG3-BMS, the antitumor effect was greatly enhanced compared to the group administered Opdivo alone. Compared with the tumor weight after the end of the administration, the OPDIVO combined with LAG3Q817-CA108.1 has better tumor inhibition effect than the OPDIVO combined with LAG 3-BMS.

Sequence listing

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Claims (9)

1. An antibody or antigen-binding fragment thereof that binds lymphocyte activation gene 3(LAG3), the antibody or antigen-binding fragment thereof comprising a VL domain and a VH domain, wherein the VL domain of the antibody or antigen-binding fragment thereof comprises LCDR1 as set forth in SEQ ID NO:5, LCDR2 as set forth in SEQ ID NO:6, and LCDR3 as set forth in SEQ ID NO:11, and the VH domain of the antibody or antigen-binding fragment thereof comprises HCDR1 as set forth in SEQ ID NO:12, HCDR2 as set forth in SEQ ID NO:13, and HCDR3 as set forth in SEQ ID NO: 14.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VL domain as set forth in SEQ ID No. 3 and the antibody or antigen-binding fragment thereof comprises a VH domain as set forth in SEQ ID No. 4.

3. The antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein the antibody or antigen-binding fragment thereof comprises a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a Fab ', a F (ab') ₂ (iii) an Fv fragment.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein the antigen-binding fragment is selected from the group consisting of an scFv or a dsFv.

5. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-4 in the manufacture of a pharmaceutical composition for the treatment of cancer, wherein the cancer is colon cancer.

6. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4.

7. A cell expressing the antibody or antigen-binding fragment thereof of any one of claims 1-4.

8. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4 and a pharmaceutically acceptable carrier.

9. The composition of claim 8, further comprising an anti-PD 1 antibody or antigen-binding fragment thereof.

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